The nm23 genes encode proteins that participate in the development and differentiation of normal tissues. Nm23 proteins are also associated with the regulation of tumor metastasis. Homologs of the highly conserved protein have been characterized in numerous tissues including those from humans, mice, Drosophila, and Myxococcus xanthus. The nm23 proteins generally consist of 150 to 180 amino acid residues. All known nm23 proteins contain a leucine zipper motif and exhibit nucleoside diphosphate kinase (NDPK) activity.
Nm23 protein accumulation is coincident with the functional differentiation of multiple epitheiial tissues in the developing mouse. At the onset of organogenesis, the amount of nm23 protein is relatively low and uniform throughout the mouse embryo. The protein begins to accumulate preferentially in the first embryonic tissues to differentiate, the developing nervous system and heart. Subsequent differentiation of liver, kidney, skin, intestine, adrenal, and stomach epithelial cells is accompanied by increased nm23 protein expression (Lasko M et al (1992) Cell Growth Differ 3:873-879).
In rodent, reduced expression of the nm23 gene systems has been correlated with increased potential for tumor metastasis. A suppressive effect of nm23 on several aspects of the cancer process, including metastasis, has been demonstrated in murine melanoma cells (Leone A et al (1991) Cell 65: 25-35). The number of metastases developed in a murine melanoma subline was inversely correlated with the expression of two murine isotypes, nm23-M1 and nm23-M2 (Baba H et al (1995) Cancer Res 55:1977-1981).
In Drosophila, an nm23 homologue, abnormal wing discs (awd), is essential for normal development. Mutation or reduced expression of awd causes abnormal tissue morphology, necrosis and widespread aberrant differentiation similar to malignant progression (Rosengard AM et al (1989) Nature 342:177-180).
Two human nm23 isoforms, nm23-H1 and nm23-H2, each consist of 152 amino acid residues with Mr of 17,143 and 17,294, respectively. The isoforms have 88% sequence identity and encode polypeptides identical to the A and B chains of human erythrocyte NDPK (Gilles et al (1991) J Biol Chem 266:8784-8789). NDPK is a hexameric enzyme, with isozymes consisting of all combinations of the A and B chains (A6, A5B . . . AB5, B6). NDPK transfers a phosphoryl group between nucleoside tri- and diphosphates via a covalent phosphoenzyme intermediate. In nm23-H1 and -H2, histidine 118 is the site of this transient phosphorylation.
Human nm23-H2 protein is also identical to the c-myc purine-binding transcription factor PuF (Postel EH et al (1993) Science 261:478-480). Myc, the protein product of the c-myc proto-oncogene, is proposed to modulate the expression of genes involved in cellular proliferation, differentiation, and tumor formation. Native PuF and purified recombinant nm23-H2 bind to DNA sequences corresponding to a nuclease-hypersensitive element (NHE) in the human c-myc P1 promoter, and induce accurate c-myc transcription in vitro.
The relationship between the DNA binding, transcriptional activation, and NDPK activities of nm23 was assessed by site-directed mutagenesis of recombinant nm23-H2. Although the NDPK phosphoenzyme active site mutant H118F was inactive in NDPK assays, it displayed normal DNA binding affinity for the c-myc promoter and retained full c-myc transcriptional activity in vitro (Postel EH and Ferrone CA (1994) J Biol Chem 269:8627-8630). This suggests that the DNA binding/transcriptional activation and the NDPK activities of nm23-H2 are independent properties of the nm23 proteins which may be associated with different biological functions.
The mechanism by which nm23 affect metastasis and development is unclear. Autophosphorylation of serine has been observed in nm23, distinct from NDPK-associated histidine phosphorylation (MacDonald NJ et al (1993) J Biol Chem 268:25780-25789). A direct correlation has been observed in mice between in vivo nm23 serine phosphorylation levels and suppression of tumor metastatic potential among control and nm23-M1 transfected murine melanoma cells. The serine phosphorylation of mouse nm23 is inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation is regulated by a signal transduction pathway. No correlation was found between nm23 NDPK activity and melanoma cell metastasis, nor was NDPK activity inhibited by cAMP (MacDonald, supra).
Co-regulated expression of nm23 and mts1 (cyclin-dependent protein kinase), another tumor-suppressor gene, alters the state of tubulin polymerization in B16 melanoma cell lines (Lakshmi MS et al (1993) Anticancer Res 13:299-303). The altered tubulin polymerization is suggested to impart invasive and metastasizing properties to the cell line.
Recently, a new human nm23 isoform, DR-nm23, has been cloned from a chronic myelogenous leukemia (CML) blast crisis cell line (Venturelli D et al (1995) Proc Natl Acad Sci USA 92:7435-7439). The DR-nm23 protein consists of 168 amino acids, and has 67% and 69% sequence identity to the nm23-H1 and H2 isoforms, respectively. DR-nm23 is involved in normal hematopoiesis and, when overexpressed, may contribute to differentiation arrest, a feature of blastic transformation in CML.
The NM23 Gene Family and Cancer
Expression levels of nm23 have been monitored throughout the development and metastasis of several types of cancer. In some tumors types, an inverse correlation exists between expression of nm23 and metastasis. For instance, elevated nm23 expression in human breast cancer tumors is associated with a decrease in lymph node metastasis and with longer patient survival. The nm23 gene product may play an important role in suppressing the metastatic phenotype (Hennessy et al (1991) J Natl Cancer Inst 83: 281-285). Stahl et al ((1991) Cancer Res 51:445-449) report that metastatic breast tumors exhibit significantly reduced levels of the nm23-H1 protein relative to the nm23-H2 protein. Tokunaga Y et al ((1993) Int J Cancer 55:66-71) likewise report that expression of nm23-H1, but not nm23-H2, is inversely associated with lymph-node metastasis. Overall survival is better in patients in which nm23-H1 expression is elevated than in those in which it is lowered. Nm23-H1 therefore has value for predicting long-term survival of human breast-cancer patients.
Steeg PS et al ((1993) Breast Cancer Res Treat 25:175-187) report a significant association between reduced nm23 expression, at the RNA or protein levels, and aggressive tumor growth in human breast cancer. Decreases in nm23 expression begin prior to actual histologically identifiable invasion. Expression of human nm23-H1 cDNA in the transfected metastatic human MDA-MB-435 breast carcinoma cell line suppresses metastatic potential by 50-90%. Transfection of human breast carcinoma cell lines with a nm23-H1 expression vector restores many phenotypically normal features to these cells (Howlett AR et al (1994) J Natl Cancer Inst 86: 1838-1844).
Differences in expression levels of nm23-H1 have been reported among normal ovary tissue, benign tumors and carcinomas. The nm23-H1 protein is absent from metastatic ovarian carcinomas more often than non-metastatic carcinomas (Kapitanovic S et al (1995) Anticancer Res 1555:587-590). Tumors that do not metastasize into the lymph nodes express nm23-H1 at significantly higher levels than tumors that do metastasize into the lymph nodes. These observations suggest that the nm23-H1 protein may have an inhibitory effect on lymphatic metastasis (Viel A et al (1995) Cancer Res 55:2645-2650).
Low levels of nm23 mRNA also correlate with high metastatic potential in malignant melanomas (Florenes VA et al (1992) Cancer Res 52: 6088-91). However, patients with higher nm23 expression in metastatic tissue tend to live longer (Xerri L et al (1994) Br J Cancer 70:1224-1228).
Similar correlations between nm23 and metastasis are reported for hepatocellular carcinomas (Iizuka et al. 95), prostate cancer (Fishman et al. 94), and leukemia cell lines (Yamashiro et al. 94).
However, among other tumor types, nm23 expression has no apparent relationship to metastatic potential. Expression of nm23 even correlates directly with severity in some of these cancers. For instance, nm23 expression in various types of thyroid cancers does not appear to play a role in metastasis. The average level of nm23 gene expression in stages I through III of differentiated thyroid carcinoma is comparable to that in multinodular goiters. In advanced stages of thyroid carcinoma (stage IV and anaplastic), a 2-fold increase in nm23 expression is observed. This suggests a direct correlation of nm23 expression with rapid cell proliferation in thyroid cancer (Zou M et al (1993) Br J Cancer 68:385-388).
Yamaguchi A et al ((1993) Br J Cancer 68:1020-1024) report that no significant correlation exists between nm23-H1 expression and colorectal tumor histology, serosal invasion, lymphatic invasion, venous invasion, or lymph node metastasis. However, Zeng ZS et al ((1994) Br J Cancer 70:1025-1030) report that nm23-H1 expression increases with local colorectal tumor severity, and in liver metastases even higher levels of nm23-H1 expression are found. In addition, Zeng et al (supra) observed two immunoreactive species of nm23-H1 expressed in roughly equal proportions in 16 patients: the predicted 17 kDa form and a larger 18.5 kDa form.
High expression levels of a 19 kDa form of nm23-H1 protein in neuroblastoma are associated with advanced stage (III and IV) disease and with N-myc gene amplification (Hailat N et al (1991) J Clin Invest 88: 341-345).
Engel M et al ((1993) Int J Cancer 55:375-9) show that high levels of nm23-H1 and nm23-H2 mRNA in human squamous-cell lung carcinoma, large-cell carcinoma, sarcoma, and carcinoids are associated with poor differentiation, advanced stage tumors and poor prognosis. In human renal carcinoma cell lines and in high stage renal cancers, levels of nm23-H1 and nm23-H2 mRNAs were elevated (Kanayama H et al (1994) Int J Urol 1: 324-331).
The discovery of DR-nm23 (Venturelli D et al, supra) raises the possibility that additional nm23-like proteins present in human tissues mediate normal or abnormal cellular processes within those tissues. Along with its diagnostic and therapeutic value, the nm23 proteins have a pronounced prognostic value for those types of tumors in which a relationship between expression of nm23 and metastasis exist. Nm23 is also useful in diagnostic, prognostic and therapeutic applications for those tumor types for which levels of expression correlate with tumor severity. Therefore, the selective modulation of the expression or activity of a novel nm23-like protein may allow the successful treatment of amenable types of cancer.